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The single-stranded DNA end binding site of p53 coincides with the C-terminal regulatory region
G Selivanova1, V Iotsova, E Kiseleva
1Department of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
Abstract:
p53 is a transcription factor that binds double-stranded (ds) DNA in a sequence-specific manner. In addition, p53 can bind the ends of single-stranded (ss) DNA. We previously demonstrated that ssDNA oligonucleotides interact with the C-terminal domain of p53 and stimulate binding to internal segments of long ssDNA by the p53 core domain. Here we show that the p53 C-terminal domain can recognize staggered ss ends of dsDNA. We have mapped the binding site for ssDNA ends to residues 361-382 in human p53 using a p53 deletion mutant (p53-delta 30) lacking the 30 C-terminal amino acid residues and a series of 22mer peptides. The binding site for DNA ends coincides with a region previously implicated in regulation of sequence-specific DNA binding by the core domain. The interaction of the C-terminal regulatory domain with the ends of ssDNA or with the protruding ends of dsDNA stimulates both sequence-specific and non-specific DNA binding via the core domain. Electron microscopy demonstrated the simultaneous binding of p53 to dsDNA and a ssDNA end. These results suggest a model in which interaction of the p53 C-terminal tail with DNA ends generated after DNA damage causes activation of sequence-specific p53 DNA binding in vivo and may thus provide a molecular link between DNA damage and p53-mediated growth arrest and apoptosis.
Insights
The p53 protein
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- p53 is a crucial transcription factor involved in DNA binding.
- p53 binds double-stranded (ds) DNA sequence-specifically and single-stranded (ss) DNA ends.
- Previous studies showed C-terminal domain interaction with ssDNA stimulates core domain binding.
Purpose of the Study:
- To investigate the p53 C-terminal domain's recognition of ssDNA ends in dsDNA.
- To map the specific binding site for ssDNA ends within human p53.
- To elucidate the mechanism by which ssDNA end binding influences p53's overall DNA binding activity.
Main Methods:
- Utilized a p53 deletion mutant (p53-delta 30) lacking C-terminal residues.
- Employed a series of 22mer peptides to map binding sites.
- Performed electron microscopy to visualize p53 binding to both dsDNA and ssDNA ends simultaneously.
Main Results:
- The p53 C-terminal domain (residues 361-382) recognizes staggered ssDNA ends of dsDNA.
- This binding site overlaps with a known regulatory region for core domain DNA binding.
- Interaction with DNA ends enhances both sequence-specific and non-specific DNA binding by the p53 core domain.
Conclusions:
- p53's C-terminal tail interaction with DNA ends, generated by DNA damage, activates sequence-specific DNA binding.
- This interaction provides a molecular link between DNA damage and p53-mediated cellular responses like growth arrest and apoptosis.
- The findings reveal a novel regulatory mechanism for p53's function in DNA damage response.